The sexual cycle of Toxoplasma gondii is limited to the feline intestine where millions of oocysts are formed and subsequently excreted in their feces. The goal of this proposal is to recapitulate the T. gondii sexual cycle in tissue culture. Towards this goal, we have already developed a protocol to harvest and culture polarized feline intestinal epithelial cells. We have also already engineered T. gondii parasites to express a negative selectable marker from a promoter specific for the asexual stage called tachyzoite. Addition of the negative selection will inhibit the rapidly replicating tachyzoites from lysing the intestinal cells, which will give the sexual stages time to develop. For this R21, we will culture these genetically manipulated parasites in our polarized feline intestinal epithetical cells as wel as develop new markers for gametes, diploid formation and meiosis to determine the extent of sexual progress. Our other goal for this R21 is to define which immortal cat cells best recapitulate sexual development so that they can be a resource for the T. gondii community. We have already immortalized our feline intestinal epithelial cells with SV-40 T-antigen and we will examine their ability to support T. gondii sexual development. We have also begun a collaboration with Jason Spence at the University of Michigan to create cat intestinal organoids in tissue culture. Creation of ex vivo sexual development conditions will allow the contribution of specific genes to biological processes and redundant pathways to be rapidly analyzed. It will also allow for a molecular analysis of the complete lifecycle of a protistan parasite, as T. gondii asexual development can already be performed in tissue culture.